Rubella virus (RUB), the sole member of the Rubivirus genus in the Togavirus family of RNA viruses, is a major human pathogen that is the most potent teratogen of an infectious nature. Use of live, attenuated vaccines control rubella, however these vaccines have been associated with arthritis in adult women and placental transfer to the fetus, contraindicating vaccination during pregnancy. The proposed research will continue a long-term study of the molecular biology of RUB aimed at understanding virus pathogenesis and evolution. The focus of study is the RUB nonstructural proteins (NSP's) involved in virus RNA replication. The NSP's have been implicated in RUB-induced pathogenesis and are of evolutionary interest in that, by computer alignment, they are more closely related to hepatitis E virus (HEV, an unclassified enteric virus) than to the alphaviruses, the other Togavirus genus. The first specific aim is to study domains of the RUB NSP's that are unique to RUB. The structure of two Zn binding domains within the protease that processes the NSP's from a precursor will be analyzed (hypothesis: the RUB protease is a metalloprotease, a type of protease not previously found in RNA viruses). Another putative Zn binding motif in the RUB NSP's will be analyzed (hypothesis: the Zn domain participates in methyltransferase activity). Finally, the function of a recently discovered domain that can be complemented by the virus capsid gene will be studied (hypothesis: complementation is at the RNA level). In the second specific aim, the effect of precursor processing on the structure of the NSP complex will be analyzed. The NSP's of plus-strand RNA viruses are multifunctional proteins and the impact of processing on these proteins, which function in a complex, is not understood. RUB produces two NSP's by a single cleavage of the precursor (other viruses produce four or more NSP's) and advantage will be taken of this minimal number of proteins to analyze the effect of cleavage on subsequent interaction of the NSP's. Binding sites between the NSP's will be mapped and the effect of independent expression and insertion of additional cleavage sites will be explored (hypothesis: complex formation is not dependent on initial synthesis as a precursor). Additionally, in vitro expression of the HEV NSP's will be used to determine how many NSP's are produced (hypothesis: HEV produces two NSP's confirming relatedness with RUB previously indicated only by computer). [unreadable] [unreadable]